Portions of this chapter are modified from: Butler, M.T. and Wallingford, J.B. Control
of vertebrate core PCP protein localization and dynamics by Prickle2. Dev. (2015). 32
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2.1: SPECIFIC PCP PROTEINS ASYMMETRICALLY PATTERN XENOPUS MULTICILIATED CELLS
The surface of tailbud stages Xenopus embryos is populated by dozens of multiciliated cells (MCCs) which drive polarized fluid flow across the surface of the tissue (Figure 7), and a role for PCP proteins in oriented multiciliary beating was first described here, in the Xenopus epidermis (Mitchell et al., 2009; Park et al., 2008). Surprisingly, there had not yet been reports of asymmetric localization of PCP proteins at the cell cortex in these cells or the tissue at large, though such localization was reported for both mouse airway (Vladar et al., 2012) and ependymal MCCs (Guirao et al., 2010). As Xenopus is a powerful tool for rapid localization screens in vivo and for studies of PCP signaling, I set out to identify faithful reporters of molecular PCP patterning in this tissue.
Several examples of cell- and tissue-specific differences in protein localization and function have been observed for different vertebrate PCP family members in the context of multiciliated epithelia. For example, Dvl2 is present at the basal bodies of MCCs, but does not display cortical asymmetry, while Dvl1 and Dvl3 are not at basal bodies, but rather are present at planar-polarized cortical crescents (Park et al., 2008; Vladar et al., 2012). The increased complexity seen in vertebrate PCP warranted further study of individual core PCP family members and challenging any assumed redundancy in their activity. To ensure adequate
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Figure 7: Multiciliated cells drive polarized flow across the Xenopus epidermis. (A) Xenopus laevis
embryo with flow and anatomical directionality denoted along with a confocal slice showing different cell types visualized via memRFP in a mosaically labeled epidermis. MCC, multiciliated cell. Note all images for Chapter 2 (Figures 7-24) are presented in this orienation. Scale bar: 50 µm
coverage over several members of the PCP multi-gene families, the localization of a wide range of GFP fusions to Xenopus PCP proteins were surveyed using mosaic expression for accurate assessment of asymmetric localization. While many previously studied Xenopus core PCP proteins localize symmetrically around the cell cortex, including Dvl2, Dvl3, Fzd7, Fzd8, and Vangl2, others, such as Fzd6, Dvl1, Pk2, and Vangl1 displayed striking asymmetric localization (Figures 8 and 9).
In Xenopus embryos, fluid flow is directed across the epidermis from the dorsoanterior to ventroposterior direction (Figure 7A); accordingly, punctate accumulations of Dvl1-GFP that were restricted to the dorsoanterior apical cell cortex of MCCs are found here, corresponding to asymmetry in the direction upstream of flow (Figure 9A). Mucociliary epithelia are comprised of two principal cell types, MCCs and mucus-secreting goblet cells, and because the MCCs are separated from one another by intervening goblet cells (Figure 7A), PCP signaling must be transmitted evenly across both cell types. It is notable then, that asymmetric accumulations of Dvl1 also occurred in goblet cells (Fig. 9B). Comparable asymmetry appeared to occur in other intercalating cell types, such as ioncytes (Dubaissi and Papalopulu, 2011; Quigley et al., 2011) and small secretory cells (Dubaissi et al., 2014; Walentek et al., 2014), which were identifiable by their significantly smaller surface area and lack of cilia in regions where only intercalating cells had been labeled.
Typically, asymmetric Dvl co-accumulates with asymmetric Frizzled at the cell cortex (Seifert and Mlodzik, 2007), and indeed, Fzd6-GFP also localized to the dorsoanterior cell cortex (Figure 8A). In most planar polarized tissues, domains enriched for Dvl and Fzd are mirrored by complementary accumulations of Prickle and Vangl (Seifert and Mlodzik, 2007), and I observed asymmetric accumulations of GFP-Pk2 (Figure 9C,D) and GFP-Vangl1 (Figure 9E,F) at the ventroposterior cell cortex in both MCCs and goblet cells as well. Moreover, co-expression of
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Figure 8: Cell localization can differ betwen particular PCP family members. (A-F’)Mosaically labeled cells in stage 31 embryos labeled with PCP proteins fused to GFP and membraneRFP and surrounded by unlabeled neighbors. Frizzled-6 (A) displays asymmetric localization while Frizzled-7 (B), Frizzled-8
(C), Dishevelled-2 (D), Dishevelled-3 (E), and Vangl2 (F) all decorate the cortex in a symmetric fashion.
Dvl1-GFP and RFP-Pk2 revealed mutually exclusive domains of enrichment (Figure 10A), and clear colocalization was observed for RFP-Pk2 and GFP-Vangl1 proteins (Figure 10B). These observations of a Fzd family member on the anterior cell face and Vangl at the posterior are consistent with previous functional studies of domineering non-autonomy of PCP signaling in this tissue (Mitchell et al., 2009), though on the other hand, it was surprising that their orientation relative to the direction of ciliary beating is reversed compared to that of MCCs in the mouse trachea (Vladar et al., 2012).
2.2: DYNAMICS OF THE ASYMMETRIC LOCALIZATION OF PK2, VANGL1, AND DVL1
Having identified useful reporters for PCP patterning in the Xenopus embryonic epidermis, I next sought to characterize the developmental dynamics of core PCP protein localization here. MCCs are derived from a basal layer of progenitor cells and insert into the mucociliary epithelium at the early tailbud stage (~St. 22), after which cilia are assembled and polarization of ciliary beating is established progressively over the next several hours (until roughly St. 30) (Billett and Gould, 1971; Drysdale and Elinson, 1992; König and Hausen, 1993). Prior to the insertion of MCCs, Dvl1-GFP decorated the apicolateral regions of goblet cells symmetrically and in a punctate fashion (Figure 11A). During tailbud stages, as ciliogenesis is completed and the refinement of ciliary orientation begins, Dvl1-GFP asymmetry is present, though the degree and coordination of asymmetry is variable at this time. Dvl1-GFP asymmetry finally reaches a maximal level in both goblet cells and MCCs across the tissue around stage 30 (Figure 11B), a time at which ciliary basal body orientations are locked in place and flow has strengthened across the epithelium (König and Hausen, 1993; Mitchell et al., 2007; Werner et al., 2007). GFP-Pk2 displayed similar dynamics to Dvl1-GFP, with asymmetric accumulations also
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Figure 9: Dvl1, Pk2, and Vangl1 asymmetrically align with Xenopus MCC flow patterns. (A-F’) MCCs (A,C,E) and goblet cells (B,D,F) in St.31 embryo surrounded by unlabeled neighbors display
asymmetric core PCP protein localization in the direction indicated by the overlying arrows. Scale bar = 5 µm
Figure 10: Pk2 forms mutually exclusive domains with Dvl1 and colocalizes with Vangl1. (A-B) Groups
of Xenopus epidermal cells labeled with RFP-Pk2 and either Dvl1-GFP (A) or GFP-Vangl1 (B). Scale bar = 10 microns
apparent by stage 24-25 that reach a maximum around stage 30 (Figure 11C,D), though with some notable differences. First, although GFP-Pk2 also localizes symmetrically to apical accumulations just prior to ciliogenesis, the punctate pattern observed for Dvl1-GFP was less prominent for GFP-Pk2 (Figure 11C). Another interesting dissimilarity was the localization of GFP-Pk2 near ciliary basal bodies labeled with Centrin-RFP at later stages (Figure 12). For the transmembrane protein Vangl1, localization of GFP fusions at early stages consisted primarily of cytoplasmic puncta, and labeling at the cell cortex was weak and diffuse at these stages (Figure 11E). This pattern may reflect the vesicular transport proteins known to be important for the processing and trafficking of Vangl proteins (Guo et al., 2013; Merte et al., 2009; Yin et al., 2012). Both the cortical localization and asymmetry of these accumulations increased as development proceeded, with a timeframe similar to that for Dvl1 and Pk2 (Figure 11F).
In efforts to chart the changes in localization asymmetry these markers over time, I quantified these dynamic localization patterns using the relative level of reporter fluorescence intensity at dorsoanterior and ventroposterior cell cortices, and mosaic labeling allowed us score cells abutting unlabeled neighbors (Figure 13). This metric demonstrates the degree to which asymmetry increased over time during PCP patterning for all three reporters examined (Figure 11G-I). Interestingly, PCP enrichment increased along with increased coordination of fluid flow (König and Hausen, 1993), and asymmetries were not easily detectable at times corresponding to stages of initial MCC intercalations. Once MCCs have intercalated and expanded their apical surface, basal bodies are docked and serve as the foundations for cilia formation. Once these cilia are visibly projecting from the apical surface, the surrounding goblet cells can be seen exhibiting asymmetric patterns that are readily detectible by eye; though no such patterns are easily discernible in the MCCs. This result suggests that the initial rotational polarity of basal bodies that is in place at the onset of ciliogenesis, which clearly depends upon intact Dvl2
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Figure 11: PCP asymmetry is refined over time, coinciding with polarized ciliary orientation. (A-D)
Patches of cells mosaically labeled with either Dvl1-GFP (A,B) or GFP-Pk2 (C,D) at stages prior to ciliogenesis (St.19) and after basal body refinements (St.31). (E,F) Patches of cells mosaically labeled with GFP-Vangl1 at stages during ciliogenesis (St.23) and during basal body refinement (St.27). (G-I) Quantifications of PCP Enrichment at different developmental stages show increasing asymmetry develops during basal body refinement. Each mark represents the enrichment value for a single cell. (G) Dvl1-GFP enrichment was measured at St.19 (n = 58), St.24 (n = 25), and St.31 (n = 321). (H) GFP-Pk2 enrichment was also measured at St.19 (n = 58), St.24 (n = 25), and St.31 (n = 321). (I) GFP-Vangl1 enrichment was measured in a narrower window at St. 23 (n = 61), St.24 (n = 268), and St.27+ (n = 465). All comparisons within graphs are highly significant (P<0.0001) except for a modest increase between Dvl1 St.24 to St.31 (P=0.0366). Error bars indicate Standard Deviation of the mean; Mann-Whitney Statistical tests. Scale bars: 10 µm.
Figure 12: Pk2 localizes to ciliary basal bodies of Xenopus MCCs. GFP-Pk2 assumes a polarized
localization near basal bodies labeled with Centrin-RFP after the finalization of basal orientation refinement (St.35 shown). Note that this cell is surrounded by similarly labeled cells, making the Pk2 cortical asymmetry less apparent. Box in (A) demarcates the area magnified in (B). Scale bar: 10μm.
Figure 13: PCP enrichment is a measure of asymmetric localization. (A) Equation used and an example
for calculating PCP Enrichment (GFP-Pk2 and memRFP shown). (B) Plot of enrichment values for dorsoanterior and the complementary ventroposterior memRFP measurements in consideration without the associated GFP PCP fusion measurements for all PCP Enrichment measurements in presented in Figures 11, 14, 15, 16, 17, 19 & 21 (n = 5,394). A value of 0 represents no difference in intensity.
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function (Mitchell et al., 2007), may not be reliant upon intracellular PCP asymmetry, but rather the surrounding tissue imparts that directional information instead. However, it has also been reported that polarity information can be relayed in the absence of detectible asymmetry in the fly wing (Strutt and Strutt, 2007, Chien et al. 2015), so perhaps asymmetric stability or activity in the absence of clear asymmetric localization may be present in this context as well.
2.3: INTERPLAY BETWEEN THE CORTICAL ASYMMETRIC LOCALIZATIONS OF PK2 AND DVL1
The interplay among Pk2 and Dvl1 during asymmetric localization was the next focus of study, as antagonistic interactions between proteins homologous to them are fundamental to amplifying visual intracellular asymmetry in other contexts (Strutt and Strutt, 2007), yet have never been examined for these particular family members or in this tissue. I first determined the effect of the well-characterized and PCP-specific dominant negative, Dvl2-DPDZpartial (Xdd1) (Sokol, 1996), shown to negatively affect PCP signaling in a variety of contexts, including in MCCs (Park et al., 2008; Wallingford et al., 2000). Expression of Dvl2-DPDZpartial significantly disrupted the normal ventroposterior restriction of Pk2-GFP (Figure 14A-C), demonstrating asymmetric Pk2 localization is dependent upon the ability of the cell to adopt a planar-polarized state. I then performed the complementary experiment by reducing Pk2 levels using an antisense morpholino (Pk2-MO1) that disrupts splicing of Pk2 (Figure 15A). While discrete crescents of Dvl1-GFP accumulation were normally oriented in the dorsoanterior direction in controls (Figure 14D), Pk2 knockdown significantly reduced this asymmetric enrichment (Figure 14E-F), and a second morpholino targeting an alternate splicing sequence provided similar results (Figure 15B). Together with the observed progressive asymmetric localization (Figure 11), these data demonstrate the efficacy and veracity of our GFP reporters
Figure 14: Intact PCP signaling is required for the formation of asymmetric core PCP complexes. (A,B’)
Mosaically-labeled epidermal cells in St.31 X.laevis embryos have GFP-Pk2 localized asymmetrically in the control situation (A) and symmetrically upon overexpression of Dvl2-ΔPDZpartial (B). (C)
Quantification of PCP Enrichment shows in that comparison to controls (n = 584), there is a significant shift upon Dvl2-ΔPDZpartial expression (n = 326, P<0.0001). (D-E’) Mosaically-labeled epidermal cells in St.31 X.laevis embryos have GFP-Pk2 localized asymmetrically in the control situation (D) and symmetrically upon Pk2-MO knockdown (E). (F) Quantification of PCP Enrichment shows that in comparison to controls (n = 508), there is a significant shift upon Pk2-MO knockdown (n = 210,
P<0.0001). Error bars indicate Standard Deviation of the mean; Mann-Whitney Statistical tests. Scale
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Figure 15: Pk2 morpholinos reduce Pk2 mRNA levels (A) RT-PCR results for the amplification of a control, ornithine decarboxylase (ODC), and morpholino-targeted Pk2 sequence from cDNA of embryos that were either uninjected or injected with Pk2 morpholino #1 or #2 into 4 of 4 cells, demonstrating a significant reduction of only the Pk2 PCR product in morpholino-injected embryos. (B) Cells in St.31 embryos labeled with PCP proteins fused to GFP and memRFP with an included dose of Pk2 morpholino #2, showing similar effects to Pk2 morpholino #1 shown in Figures 12 and 16. (C) Graph depicting changes in normal localization of Vangl1 (n = 519) and Dvl1 (n = 508) caused by Pk2 knockdown with a second Pk2 morpholino (Pk2-MO#2) targeting an alternative splicing site from the first. PCP enrichment is significantly reduced for both GFP-Vangl1 (n = 137, P<0.0001) and Dvl1-GFP (n = 131, P<0.0001) in morphant cells. Error bars indicate Standard Deviation of the mean, Mann-Whitney statistical tests. Scale bars: 10μm.
for core PCP protein localization in this tissue and tools for manipulating PCP signaling in this system.
2.4: ROLE OF PCP EFFECTORS INTURNED AND WDPCP IN PK2 PROTEIN LOCALIZATION
With useful PCP reporters in hand, I sought to uncover novel reporters to address outstanding questions in vertebrate PCP signaling. First, I assessed the role of the “PCP Effector” proteins in the patterning of polarity complexes, as the role for these proteins in PCP signaling remain contentious. First identified as planar polarity proteins by genetic screens in Drosophila (Collier et al., 2005; Gubb and Garcia-Bellido, 1982), Inturned and Fritz were placed genetically downstream of core PCP protein function (Collier et al., 2005; Lee and Adler, 2002; Wong and Adler, 1993), though a more recent study suggests that Fritz overexpression can influence core PCP protein localization in Drosophila (Wang et al., 2014). Curiously, the vertebrate orthologues, (called Intu and Wdpcp/Fritz) were found to control ciliogenesis, first in the Xenopus epidermis (Kim et al., 2010; Park et al., 2006) and later in mice (Cui et al., 2013; Zeng et al., 2010). Intu apparently plays only a modest role in PCP mediated processes such as convergent extension (Park et al., 2006), while Wdpcp is essential for both convergent extension in Xenopus (Kim et al., 2010) and planar polarization of cochlear hair cells in the mouse (Cui et al., 2013). I performed knockdown of Wdpcp and Intu using morpholinos whose action has been validated by genetic studies in mice (Cui et al., 2013; Kim et al., 2010; Park et al., 2006; Zeng et al., 2010). Wdpcp knockdown strongly disrupted the planar polarized localization of Pk2, while Intu knockdown had a far more modest, though still significant effect (Figure 16A- C). These data reveal an important role for Wdpcp in control of core PCP protein asymmetry in vertebrates, the mechanism of which will be important to determine.
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Figure 16: Wdpcp knockdown disrupts core PCP patterning. (A-B’) Mosaically-labeled epidermal cells
in St.31 X.laevis embryos have GFP-Pk2 localized asymmetrically upon Intu-MO knockdown (A) and symmetrically upon Wdpcp-MO knockdown (B). (C) Quantification of PCP Enrichment shows that in comparison to controls (n = 64), there is a significant shift upon Intu-MO knockdown (n =187, P=0.0031) and more significant shift upon Wdpcp-MO knockdown (n =137, P<0.0001). Error bars indicate Standard Deviation of the mean; Mann-Whitney statistical tests. Scale bars: 10 µm.
2.5: ROLE OF PK2 IN THE CONTROL OF ASYMMETRIC CORTICAL VANGL1 DYNAMICS
The next question I chose to address concerns the interplay between Pk2 and Vangl1. In Drosophila, Pk physically interacts with and clusters Vang at the apicolateral membrane, and this behavior promotes Vang accumulation on the proximal side of cells in the wing epithelium (Bastock et al., 2003; Jenny et al., 2003). Though Vangl2 and Pk1 are implicated in vertebrate PCP (Liu et al., 2014; Song et al., 2010; Takeuchi et al., 2003; Torban et al., 2008; Vladar et al., 2012), Vangl1 and Pk2 remain relatively seldom studied, and it is unknown in vertebrates if either Pk protein is required for normal localization of either Vangl protein. I found that Pk2 knockdown eliminated the asymmetric accumulation of GFP-Vangl1 at the ventroposterior cell cortex (Figure 17A,B), while conversely, Pk2 overexpression increased Vangl1 apicolateral enrichment to such a degree that accumulations were no longer as restricted to just ventroposterior regions, but rather enriched in other areas around the cell periphery (Figure 17C). Additional analysis confirms that Pk2 knockdown disrupts asymmetry by suppressing the enrichment of Vangl1 at the cortical regions of the cell (Figure 18A,B), and together, these observations are consistent with a previously defined role for Pk in the clustering of Vang (Bastock et al., 2003; Cho et al., 2015; Strutt and Strutt, 2007). I conclude that Pk2 promotes cortical Vangl1 concentration, while an excess of Pk2 promotes further GFP-Vangll enrichment that becomes increasingly unrestricted from the ventroposterior cell face.
In the reciprocal experiment, overexpression of Vangl1 showed a clear reduction in the planar-polarized, cortical accumulation of GFP-Pk2 (Figure 19A,C), while enrichments of Dvl1- GFP were still present yet not asymmetrically polarized (Figure 19B,C). In light of previous evidence that Vang participates in the control of Pk protein levels (Strutt et al., 2013b), our
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Figure 17: Pk2 expression levels influence the asymmetric localization of Vangl1. (A-C’) Mosaically
labeled epidermal cells in St.31 X. laevis embryos have GFP-Vangl1 localized asymmetrically in the control situation (A), whereas it is absent from apicolateral enrichments upon Pk2-MO knockdown (B) and localized more symmetrically with Pk2 overexpression (C). (D) Quantification of PCP Enrichment shows that in comparison to controls (n = 519), there is a significant shift upon Pk2-MO knockdown (n = 355, P<0.0001) and Pk2 overexpression (n = 217, P=0.0002). Error bars in D indicate Standard Deviation of the mean; Mann-Whitney Statistical tests. Scale bars: 10 µm.
Figure 18: Pk2 influences the apicolateral enrichment of Vangl1. (A) Schematic of quantification
method of data presented in (B,C). Either the average cortical fluorescence intensity or the average intensity of the remaining central area within each cell was divided by the average fluorescence intensity of the total apical surface area (both regions combined). (B) Quantification data showing a significant loss of cortical enrichment of GFP-Vangl1 upon Pk2-MO knockdown. Each dot represents one cell, and each cell has both a Central and a Cortical quotient presented with the total apical fluorescence measure as the divisor. Combined, these two measurements are highly significant from another in the control (n = 123) and Pk2-OE (n = 63) conditions (P<0.0001) but not significant for Pk2-MO#1 knockdown (n = 94,
P=0.2854). Error bars indicate Standard Deviation of the mean; Mann-Whitney statistical tests. (C)
Quantification data showing a significant loss of cortical enrichment of GFP-Vangl1 upon
overexpression of Pk2-ΔPETΔLIM using the same quantification scheme as (B). Combined, these two measurements are highly significant from another in the Pk2-ΔPET (n = 85, P<0.0001)) and Pk2-ΔC2 (n = 84, P<0.0001)) conditions but not significant for Pk2-ΔPETΔLIM (n =58, P=0.1519). Error bars indicate Standard Deviation of the mean; Mann-Whitney statistical tests. Scale bar: 10 μm.
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Figure 19: Vangl1 Overexpression leads to PCP patterning defects. (A) Cells in stage 31 embryos
labeled with PCP proteins fused to GFP and membraneRFP as well as overexpressing Vangl1 display loss of Dvl1-GFP asymmetry and reduction in GFP-Pk2 cortical enrichment. (B) Graph depicting changes in PCP Enrichment of Pk2 and Dvl1 caused by Vangl1 overexpression (V1-OE). PCP
enrichment is significantly reduced in cells overexpressing Vangl1 (n = 60 for Pk2, n = 55 for Dvl1) in comparison to controls for both GFP-Pk2 (n = 64) (p<0.0001***) and Dvl1-GFP (n =243)
(p<0.0001***). (C) Quantification data showing a significant loss of cortical enrichment of GFP-Pk2 but not Dvl1-GFP upon Vangl1 overexpression (V1-OE). Each dot represents one cell, and each cell has both a Central and a Cortical measurement presented as divided by the total apical fluorescence (See Fig. 18A for quantification schematic). These Central and Cortical measurements are highly significant from one another in both control Pk (n = 33, P<0.0001) and Dvl1 (n = 36, P<0.0001), Dvl1 with V1-OE (n = 53, P<0.0001), and Pk2 with V1-OE (n = 53, P=0.0002) conditions, but only in the case of GFP-Pk2 in V1-OE embryos is the mean Cortical enrichment less than the Central measure. Error bars indicate Standard Deviation of the mean; Mann-Whitney statistical tests. Scale = 10 µm.
results suggest that Vangl1 overexpression may be leading to increased levels of Pk2 degradation, which in turn leads to a reduction in Pk2 and associated Dvl1 patterning defects.
Given the demonstrated effects of Pk2 on the asymmetric enrichment of Vangl1, I next asked if Pk2 influences the dynamics of Vangl1 turnover at the cell cortex. Indeed, asymmetric enrichment of Fzd is driven in part by differences in Fzd turnover at distinct locations along the